For a number of years, immunoassay procedures have replaced other procedures used for in vitro diagnostic methods to detect or quantitate a variety of antigens and/or antibodies in fluids and particularly body fluids such as blood serum or urine with important biologic or pharmacologic properties. The high levels of sensitivity and specificity achieved with immunoassays result from the specific, high-affinity, reversible binding of antigens to antibodies, and from the existence of methods for attachment of sensitive detectable labels (radioactive isotopes, fluorophores, ferritin, free radicals, bacteriophages and enzymes) to antibodies or antigens. Radioactive isotopes and enzymes are currently the most extensively used labels although the use of enzymes is generally preferred and the number of sensitive, specific immunoassays employing enzyme "tags" is expanding rapidly.
Immunoassay techniques are based upon the complex binding of the antigenic substance being assayed with an antibody or antibodies in which one or the other member of the complex may be labeled, permitting the detection and/or quantitative analysis of the target substance by virtue of the label activity associated with the labeled antigen complex or antibody. Immunoassays are generally classified into two groups: the heterogeneous immunoassay in which a labeled antigen or antibody is separated from the labeled antigen-antibody complex before measurement of label activity in either fraction; and the homogeneous immunoassay in which the activity of labeled antigen is measured in the presence of labeled antigen-antibody complex.
Two such diagnostic assay techniques used to determine the presence or amount of antigen in body fluids are generally known as "competitive" assays and "non-competitive or sandwich" assays. Typically, in "competitive" assay techniques, an unlabeled polyclonal antibody preparation bound to a solid support or carrier is first reacted with a labeled antigen reagent solution and then with the body fluid sample wherein the antigen in the sample competes with the labeled antigen for sites on the supported antibody. The amount of labeled antigen reagent displaced indicates the quantity of antigen present in the fluid sample to be detected.
In the case of a "sandwich" or "non-competitive" assay, a quantity of unlabeled polyclonal or monoclonal antibody bound to a solid-support or carrier surface, is reacted with a body fluid sample being evaluated for antigens, and then, after suitable incubation time and washing, the sample is further incubated with a solution of labeled antibody. The labeled antibody bound to the solid phase in an antibody-antigen-antibody sandwich or the amount of unbound labelled antibody in the liquid phase would be determined as a measure of the presence of antigen in the test sample.
The ready availability of monoclonal antibodies has made possible the modification of immunoassay procedures such as disclosed, for example, in U.S. Pat. Nos. 4,376,110 and 4,486,530 to David et al., wherein antibodies specific to antigens could be employed to detect such antigens in body fluids reducing or eliminating certain intermediate steps in the assay. Nonetheless, the processes heretofore typically employed require measured quantities of reagents and controlled extended reaction times as well as several washings and quenching, limiting these procedures to hospitals and laboratories where trained personnel and suitable equipment are available to perform the assays. Thus, the desirability of a relatively simple procedure and apparatus which made readily possible such assays in the physicians office or even their use by lay persons in home health care programs would be evident.
Over the years, many attempts have been made to develop more accurate, sensitive and definitive tests and devices for immunoassay diagnostic testing. For example, in U.S. Pat. No. 4,168,146 to Grubb et al., U.S. Pat. No. 4,200,690 to Root et al., and U.S. Pat. No. 4,373,932 to Gribnau et al., immunoassays are described for the detection of the presence of various antigens in body fluids and the like which employ reagents such as antibodies bound to a variety of porous support or carrier materials and dye labeled antibodies or the like. Such devices and methods, however, are generally directed to immunoassays for specific antigenic substances using particular types of porous carrier materials and labels for the antibody reagents which exhibit limited sensitivity and accuracy while requiring several operations including washing and/or quenching steps and extended periods for incubation which limit their suitability for use by less skilled personnel in a doctors office or home care situation.
More recently, there have been disclosed, for example, in U.S. Pat. Nos. 4,632,901 and 4,727,019 to Valkirs et al.; U.S. Pat. No. 4,639,419 to Olson et al.; U.S. Pat. No. 4,703,017 to Campbell et al.; and U.S. Pat. No. 4,786,589 to Rounds, procedures for conducting particular types of immunoassays in a relatively short period of time and routine fashion which employ generally simple apparatus. However, while such procedures and devices reduce the complexity and time for certain assays, several steps including a washing and/or quenching step are still generally necessary, the sensitivities thereof are limited and the test specimens are generally not suitable for extended periods of storage. Thus, even further simplification or efficacy would be desirable.